We have been studying three regulatory elements of the human c-myc proto- oncogene and the proteins which bind to them. 1) 1.5 kb upstream of promoter P1 resides a cell-type and differentiation specific positive cis-element. A novel protein binding to this element has been purified and subjected to proteolytic degradation and partial sequence determination. The information thus obtained allowed the cloning of a gene encoding a novel DNA binding protein. This protein possesses a structure comprised of alternating amphiphathic helices (five) and repeating units (four). The minimal sequence specific DNA binding domain is composed of two helices and two repeats. Analysis of the expression of this factor reveal it to be tissue specific and highly regulated. Functional characterization of the protein is ongoing. 2) 100-150 bp upstream of P1 a complex set of trans-factors binds to a cytidine-rich element repeated five times. Some of the factors which interact with this element possess the ability to recognize specific single-stranded sequences. One of the pyrimidine-rich strand binding proteins is hnRNP protein K. Importantly, the activity of the cis- element in vitro can be reduced by inclusion of specific single- stranded oligonucleotide competitors thereby indicating the potential for a molecule with hnRNP K-like properties to regulate c-myc. Additional proteins interact with the purine strand. the purine strand binding factors possess several unusual properties which correlate well with the activity of this element in in vitro transcription systems. The purification of these factors with an aim of cloning their genes is in progress. 3) Approximately 1 kb downstream of c-myc promoter P1 is an element which has been found to be mutated frequently in Burkitt lymphoma. Several proteins have been shown by cross-linking or binding with SDS-PAGE purified and renatured polypeptides to interact with this element. Some of these proteins possess unusual properties which suggest interesting regulatory mechanisms for controlling c-myc expression. The purification and characterization of these proteins as well as functional characterization of their binding sites are the topics of current studies.